EP2702949B1 - Sampling device and system for capturing biological samples in a bodily fluid, and method of manufacturing same - Google Patents

Description

The present invention relates to a sampling device adapted to be inserted into a hollow needle-like tubular tip or catheter and to open out of the tip for contact with a body fluid containing biological targets to be taken, a sampling system incorporating this tip and this device which is slidably mounted thereon, and a method of manufacturing this device. The invention applies to samples made in particular in vivo in body fluids of the human body, for example circulating body fluids, and in particular the blood, the cerebrospinal fluid, the interstitial fluid or the lymph, these fluids possibly containing as targets of proteins, oligonucleotides such as RNA or DNA, antibodies, enzymes or cells, without limitation.

For many years, analytical techniques, whether based on genomics, proteomics or immunology, have been progressing and reaching remarkable levels of sensitivity. These techniques are based on the recognition of elements of interest or biological targets that must be extracted from the other elements present in the sample taken whether it is done in vivo or ex vivo. If the target is absent or too small compared to the sensitivity of the analysis method, then the measurement will not be possible.

The sample preparation methods aim at capturing the targeted targets and bringing them into contact after concentration with a functionalized surface on which a measurement is made. They are very interesting because they allow, by concentration of this target, to release the constraint on the measurement sensitivity. On the other hand, they are inoperative if the target is not present in the sample taken. This pitfall is evident for example, during the blood test that saw the race to the reduction of the sampled volume is jeopardized by the presence or absence of the desired element and by the sensitivity of the measuring system.

A conventional method is to mix nanoparticles carrying recognition sites, such as nanobeads, with the sample containing the target and then perform volume recognition and finally recover these nanoparticles by centrifugation or magnetic attraction before performing a controlled re-release of targets captured on a measurement surface.

Another known method is to carry out a recirculation of the liquid to be tested on a surface having the recognition sites in question.

The problem of testing body fluids circulating in the human body can be understood in the same way. In the example where the volume to be tested is the whole blood or cerebrospinal fluid, it is analogous to consider the same approach which consists in injecting metallic and / or magnetic nanoparticles into the body fluid or under the skin, leaving them recognize the targets and then recover them for example by affixing a local magnetic field or by filtration on an extracorporeal bypass. This approach requires a very thorough study of the particles injected in terms of toxicity and filtration in the kidneys, liver, etc. An unsolved problem to date is a satisfactory recovery of such injected particles.

In order to overcome the problems that may arise from the injection of such nanoparticles into the human body, the solutions developed to date are generally based on the encapsulation of these metallic and / or magnetic nanoparticles. in different materials and in particular in biocompatible polymers. Depending on the porosity of this polymer, it can act as a filter, blocking biological species exceeding a certain size. By biological species, we mean here cells, molecules, viruses, bacteria or antibodies.

In known devices, the use of a coating or encapsulation improves the performance of the measuring device whether for contrast efficiency, tissue tolerance or capture ability. However, a major disadvantage of these devices is that they are not designed to recover the nanoparticles after they come into contact with the desired targets in the fluid in question, in particular for the capture of these targets or following an injection beforehand. to an MRI (Magnetic Resonance Imaging) analysis. In other words, there is the problem in these known devices the fate of nanoparticles injected in vivo .

The document FR 2 955 024 in the name of the Applicant makes it possible to remedy this drawback by presenting a device for transient contacting of target capture media to be analyzed with a body fluid containing them, this device comprising a sampling tip including a contacting end with the fluid is bonded to capture media that are covered by a biocompatible and porous crosslinked polymeric layer. This layer is designed to retain the capture media and let only particles including these targets smaller than a size limit, so that the capture media are recoverable after contacting by dissolution of this layer. This document mentions the possible integration of capture media to a mesh structure connected to the tip whose strands are covered with said layer, which encapsulates these capture media which may consist of functionalized nanoparticles. This mesh structure is folded into a guide structure for example tubular and deployed reversibly out of it during sampling. The mesh structure is intended to be deployed in the body, for example in a blood vessel, in the same way as a "stent". It is therefore clear that the biological fluid flows between the strands, that is to say between the mesh of the device.

Although the devices presented in this document provide quite satisfactory results, the Applicant has sought to optimize the adhesion or adhesion of the entire polymeric layer to such a mesh structure, so as to minimize the risk of detachment of polymeric fragments vis-à-vis the surface that this layer covers.

The document WO-A1-2010 / 145824 has a detection device for the enrichment of samples, comprising a three-dimensional detection surface which is made functional by a multitude of detection receivers that it has and which can be microstructured for example in the manner of a mesh network. This document does not relate to the adhesion to a reinforcement of a polymeric layer coating capture media separate from this reinforcement.

An object of the present invention is to provide a sampling device adapted to be inserted into a hollow tubular needle tip or catheter and to open out of the tip for contact in particular in vivo with a body fluid containing biological targets to be taken, which overcomes the aforementioned drawbacks, the device comprising a reinforcement microstructured by perforations, and a biocompatible and porous crosslinked polymeric layer, which comprises capture supports able to capture said targets and which is adapted to retain these media to the fluid and let pass only particles of the fluid including these targets smaller than a size limit.

For this purpose, a device according to the invention is such that said polymeric layer fills all or part of said openings, so as to be retained by said frame.

According to one embodiment of the invention, the armature is inscribed in at least one cylindrical surface of greater transverse dimension between 500 μm and 2 mm, the armature being embedded in said polymeric layer.

In other words, the polymeric layer fills all or part of the openings of the armature, extending on both sides of the latter.

The armature may comprise an outer face and delimit an internal volume, the polymeric layer extending through said openings of this internal volume to this outer face and beyond the latter.

The frame may be tubular, in which case it encloses a cylindrical internal volume, said polymeric layer then extending on either side of the frame. Note that this generally tubular reinforcement microstructured by openings (ie through micro-openings of dimensions of the order of one or more tens of microns) pass the non-crosslinked polymeric material (ie not yet gelled) of the porous layer so as to partially or completely fill the internal space of this frame, which significantly improves the attachment of this layer to the frame by this optimized adhesion on both radially inner and outer faces of the frame. As a result, the risks of stalling of the layer vis-à-vis the armature and thus of releasing a fragment of this layer in the bodily fluid seat of the sample are minimized.

By "tip" means in the present description a tip that may correspond to all or part of an injection needle or sampling, a catheter or an external system, which is adapted to be introduced into the medium containing the body fluid. Such a medium may for example be the vein of a human being or animal.

By "at least one cylindrical surface" is meant in the present description one or more cylindrical surfaces (ie each being defined on its height by a generatrix and on its cross section by a director in the form of any curved line, for example a director in the form of an ellipse or a circle), it being specified that the cross section of the external face of the armature may be constant (where this face is inscribed in a single cylindrical surface) or variable (where this face is successively inscribed in several cylindrical surfaces and / or in one or more frustoconical surfaces succeeding one or more cylindrical surfaces).

The crosslinked layer of the device according to the invention has a selective permeability and avoids direct contact between the capture media and the fluid to prevent an immune reaction, to prevent the diffusion of all or part of the capture media in the fluid, to selectively capture the targets according to their size and to impose less mechanical constraints on surrounding biological tissues because of its flexibility.

According to another characteristic of the invention, said armature may have a single longitudinal axis of symmetry which is intended to be parallel to that of said mouthpiece, said armature retaining a generally tubular geometry in its positions where it is inserted into the mouthpiece and where it opens out of it.

In other words and contrary to the mesh structure of the document WO-A1-2011 / 086486 aforementioned that is deployed out of the tip and that is folded into the latter, an armature according to the invention is unfit to be deployed out of the tip (ie extended or open, for example unrolled) and folded into the latter (ie retracted more compactly, for example wound) because it is substantially indeformable and has a geometry of revolution conforming to the inner face of the corresponding end region of the tip.

According to another characteristic of the invention, the crosslinked polymeric layer may have a viscosity, measured by a cone-plane rheometer, which is equal to or greater than 100 mPa.s and preferably between 150 mPa.s and 5800 mPa.s. . Beyond these values, the viscosity is too high, which limits the possibilities of shaping the polymer.

It will be noted that this particular viscosity of the polymeric layer contributes significantly to the good adhesion of this layer to the microstructured reinforcement of the device according to the invention, which is characterized by irregularities and surface roughnesses formed by the radially outer and inner edges of the micro-perforations.

Advantageously, this micro-perforated reinforcement of a device according to the invention may be of the mesh screen type, woven or braided, comprising a multitude of openings or interstices separated two by two in a step between 30 microns and 60 microns.

According to a particularly advantageous embodiment of the invention, said frame is of wire mesh type, woven or screened. It can then be substantially flat, or form a closed surface so as to form an internal volume. It may then comprise one or more micro-wire cylindrical tubes of substantially circular cross-section with, in the case of several tubes, their respective longitudinal axes of symmetry which are parallel. It can be concentric tubes.

Preferably, said reinforcement has a thickness of between 10 μm and 100 μm, and said at least one cylindrical surface has a substantially elliptical or circular section.

It will be noted, however, that a reinforcement according to the invention may furthermore have, on its outer and / or inner faces, non-perforated but microstructured zones in another manner, for example by recesses (ie non-radially through holes) and / or continuous or discontinuous reliefs of dimensions - such as the radial depth - less than 1 mm, these recesses and / or reliefs preferably having such dimensions of between 20 .mu.m and 90 .mu.m on these external and / or internal faces.

In particular, such a reinforcement microstructured by these recesses and / or reliefs in addition to said micro-openings can be obtained by sandblasting one or more areas of its initially smooth outer face (s).

According to another characteristic of the invention, said polymeric layer may form, with respect to said outer face of the armature, an outer coating substantially coaxial with this armature and having a thickness of between 50 μm and 300 μm.

According to the invention, said armature may be embedded in said polymeric layer over part of its axial length of between 1 mm and 5 cm, this polymeric layer may advantageously have a volume of between 1 ml and 10 ml.

It will be noted that such a volume makes it possible to enclose a large quantity of capture media, such as nanobeads, which is particularly useful when it is desired to capture minority species circulating in a body fluid.

According to another characteristic of the invention, said capture media may comprise magnetic or non-magnetic nanoparticles which are functionalized on the surface by grafted functions capable of capturing said targets, which have a greater transverse dimension of between 50 nm and 500 nm and which are embedded in the mass of said polymeric layer, these nanoparticles preferably being nanoshells or nanospheres with based on an iron oxide with a diameter of between 80 nm and 200 nm and functionalized by anionic or cationic functions (or alternatively by antibodies, oligonucleotides such as aptamers, chromatography-type surface functions and the functions of peptide libraries and oligonucleotides).

Advantageously, the reinforcement may be made of a metallic material, preferably surgical grade stainless steel, made of silicon or of a polymeric material such as a silicone, and the polymeric layer is based on at least one polymer biocompatible and reversible gelation selected from the group consisting of alginate gels, alginate and poly-L-lysine copolymers, chitosan, agarose, cellulose, poly (trimethylammonium ethyl acrylate methyl sulfate) -b poly (hydroxyethylmethacrylate (HEMA), poly (hydroxyethylmethacrylate-methyl ethacrylate (HEMA-MMA) and the other methacrylate-based copolymers, polyethylene glycols, acrylonitrile and polyethylene glycol copolymers, polysaccharides and their mixtures.

Even more advantageously, the armature may be provided on its surface with functional groups creating chemical bonds between the armature and the polymeric layer, preferably carboxylic acid or amine groups in the case where the armature is metallic for a connection with hydroxyl groups of this layer.

Preferably, the polymeric layer is based on at least one alginate gel which is obtained by means of polycations preferably selected from the group consisting of polycations of calcium, barium, iron and strontium. The use of an alginate is indeed particularly advantageous, because it is perfectly biocompatible, non-toxic and allows the targets to capture the cross. In addition, it is polymerizable and gellable to ambient temperatures and remains in gelled form at body temperatures and pH corresponding to physiological conditions.

Also advantageously, the crosslinked polymeric layer may have a Young's modulus, measured from compression tests carried out with a rheometer, included between 50 kPa and 270 kPa. Beyond these values, the polymer becomes difficult to shape.

Advantageously, the polymeric layer may have a porosity defining said size limit which is between 10 nm and 1 μm, with a surface porosity of between 10 nm and 50 nm and a porosity in the mass of between 100 nm and 1 μm in the preferred example of an alginate gel.

• un embout tubulaire creux de type aiguille ou cathéter qui présente un diamètre interne compris entre 500 µm et 2 mm,
• un dispositif de prélèvement inséré dans l'embout et apte à déboucher par coulissement hors d'une extrémité de cet embout en vue d'une mise en contact notamment in vivo avec un fluide corporel contenant des cibles biologiques à prélever, et
• un organe de poussée apte à faire coulisser réversiblement ledit dispositif de prélèvement hors dudit embout.

A sampling system according to the invention comprises:

• a tubular hollow tip of the needle or catheter type which has an internal diameter of between 500 μm and 2 mm,
• a sampling device inserted into the mouthpiece and able to slide out of one end of this mouthpiece for contacting, in particular in vivo, with a body fluid containing biological targets to be taken, and
• a thrust member adapted to reversibly slide said sampling device out of said tip.

This system of the invention is characterized in that this device is as defined above and is optionally provided with a connecting means at said end of the tip.

• un corps de pompe dans lequel est monté ledit embout, et
• une tige insérable dans ledit embout pour y faire coulisser ledit dispositif de prélèvement suivant une translation réversible parallèle à l'axe de symétrie de l'embout.

According to another characteristic of the invention, said thrust member may be of the syringe type, and comprises:

• a pump body in which said tip is mounted, and
• a rod insertable in said tip to slide said sampling device in a reversible translation parallel to the axis of symmetry of the tip.

Preferably, there is a liquid, liquid physiological type, between said insertable rod and said sampling device. This limits the degradation of the polymer during the push. The liquid is advantageously injectable.

1. a) préparation d'un composite polymérique non réticulé incorporant lesdits supports de capture et ladite couche polymérique les recouvrant à l'état non réticulé,
2. b) insertion de ladite armature dépourvue de ce composite dans un moule tubulaire, avec optionnellement une liaison de l'armature à une extrémité de prélèvement de l'embout (de préférence, le moule est perforé de telle sorte que la solution de gélification est en contact avec la périphérie du polymère durant la gélification de ce dernier, cela permettant de mieux maîtriser la porosité de surface ; la taille des perforations est avantageusement inférieure à 1 mm).
3. c) assemblage de l'embout contenant cette armature dans un organe de prélèvement de type seringue,
4. d) prélèvement du composite non réticulé préparé en a) par cet organe de prélèvement, pour injecter ce composite à l'intérieur de l'embout au contact de l'armature, puis
5. e) réticulation dans un bain gélifiant de l'embout qui est rempli du composite non réticulé injecté en d) et que l'on a préalablement extrait de cet organe de prélèvement, pour l'obtention de ladite couche polymérique réticulée solidaire de l'armature.

A manufacturing method according to the invention of a sampling device such as that defined above comprises the following steps:

1. a) preparing an uncrosslinked polymeric composite incorporating said capture supports and said polymeric layer covering them in the uncrosslinked state,
2. b) inserting said armature without this composite into a tubular mold, optionally with a connection of the armature to a sampling end of the mouthpiece (preferably, the mold is perforated so that the gelling solution is in contact with the periphery of the polymer during the gelation of the latter, this makes it possible to better control the surface porosity, the size of the perforations is advantageously less than 1 mm).
3. c) assembling the tip containing this armature in a syringe-type sampling device,
4. d) removal of the non-crosslinked composite prepared in a) by this sampling member, for injecting this composite inside the tip in contact with the frame, then
5. e) crosslinking in a gelling bath of the tip which is filled with the non-crosslinked composite injected in d) and which has been previously extracted from this sampling member, to obtain said crosslinked polymeric layer integral with the reinforcement .

• a1) une dispersion dans une solution tampon aqueuse desdits supports de capture comprenant des nanoparticules fonctionnalisées magnétiques ou non, puis
• a2) un ajout sous agitation à la dispersion obtenue en a1) d'au moins un polymère biocompatible et à gélification réversible, pour l'obtention du composite non réticulé dans lequel sont noyées ces nanoparticules.

Advantageously, step a) may comprise:

• a1) a dispersion in an aqueous buffer solution of said capture supports comprising magnetic or non-magnetic functionalized nanoparticles, then
• a2) a stirring addition to the dispersion obtained in a1) of at least one biocompatible and reversibly gelling polymer, for obtaining the uncrosslinked composite in which these nanoparticles are embedded.

• la figure 1 est une vue schématique illustrant différentes phases d'une méthode de prélèvement de cibles biologiques dans un fluide corporel selon un exemple de réalisation de l'invention,
• la figure 2 est une vue partielle de face d'une installation pour la préparation d'un composite polymérique réticulé inclus dans un dispositif de prélèvement selon l'invention,
• la figure 3 est une vue schématique en coupe longitudinale d'un embout de type aiguille qui contient un dispositif de prélèvement selon l'invention et que l'on a introduit dans une veine du corps humain, le dispositif étant en position rentrée dans l'embout,
• la figure 4 est une vue schématique en coupe longitudinale de l'embout de la figure 3 toujours introduit dans cette veine, mais le dispositif de prélèvement étant en position partiellement sortie par rapport à l'embout,
• la figure 5 est une vue de détail d'une armature micro-ajourée d'un dispositif de prélèvement selon un exemple de l'invention, cette armature étant dépourvue du composite polymérique réticulé destiné à la recouvrir,
• la figure 6 est une photographie montrant le recouvrement de l'armature de la figure 5 par ce composite réticulé,
• la figure 7 est une vue partielle en plan d'un embout de type aiguille contenant une armature filaire non conforme à l'invention dépourvue de ce composite réticulé,
• la figure 8 est une vue partielle en plan d'un embout de type aiguille contenant une armature micro-ajourée selon un autre exemple de l'invention dépourvue de ce composite réticulé,
• la figure 9 est une photographie montrant le recouvrement par ce composite réticulé de l'armature filaire de la figure 7, immédiatement après injection et réticulation du composite dans l'embout au contact de l'armature,
• la figure 10 est une photographie montrant le recouvrement par ce composite réticulé de l'armature micro-ajourée de la figure 8, immédiatement après injection et réticulation du composite dans l'embout au contact de l'armature,
• la figure 11 est une photographie montrant le recouvrement par ce composite réticulé de l'armature filaire de la figure 7, après lavage à l'eau du dispositif de prélèvement visible à la figure 9, et
• la figure 12 est une photographie montrant le recouvrement par ce composite réticulé de l'armature micro-ajourée de la figure 8, après lavage à l'eau du dispositif de prélèvement visible à la figure 10.

Other advantages, characteristics and details of the invention will emerge from the additional description which will follow with reference to the accompanying drawings, given solely by way of example and among which:

• the figure 1 is a schematic view illustrating different phases of a method of sampling biological targets in a body fluid according to an exemplary embodiment of the invention,
• the figure 2 is a partial front view of an installation for the preparation of a crosslinked polymeric composite included in a sampling device according to the invention,
• the figure 3 is a schematic view in longitudinal section of a needle-like tip which contains a sampling device according to the invention and which has been introduced into a vein of the human body, the device being in the retracted position in the tip,
• the figure 4 is a schematic view in longitudinal section of the tip of the figure 3 always introduced into this vein, but the sampling device being in the partially extended position relative to the tip,
• the figure 5 is a detail view of a micro-perforated reinforcement of a sampling device according to an example of the invention, this reinforcement being devoid of the crosslinked polymeric composite intended to cover it,
• the figure 6 is a photograph showing the coat of the frame of the figure 5 by this crosslinked composite,
• the figure 7 is a partial plan view of a needle tip containing a wire frame not in accordance with the invention without this crosslinked composite,
• the figure 8 is a partial plan view of a needle-like tip containing a micro-perforated reinforcement according to another example of the invention devoid of this crosslinked composite,
• the figure 9 is a photograph showing the recovery by this crosslinked composite of the wire frame of the figure 7 , immediately after injection and crosslinking of the composite in the tip in contact with the reinforcement,
• the figure 10 is a photograph showing the recovery by this crosslinked composite of the micro-openwork frame of the figure 8 , immediately after injection and crosslinking of the composite in the tip in contact with the reinforcement,
• the figure 11 is a photograph showing the recovery by this crosslinked composite of the wire frame of the figure 7 after washing with water the sampling device visible at the figure 9 , and
• the figure 12 is a photograph showing the recovery by this crosslinked composite of the micro-openwork frame of the figure 8 after washing with water the sampling device visible at the figure 10 .

In the sampling method illustrated in figure 1 in a first step A, an incubation is carried out in a fluid containing targets 1 (eg proteins) of a crosslinked (ie gelled) polymer composite 2 constituted in this example by a layer 2a of a gel calcium alginate in which are embedded capture media 2b formed of magnetic nanoparticles advantageously based on Fe ₂ O ₃ (this composite 2 is integral with a micro-perforated reinforcement according to the invention, not shown here and described hereinafter below with reference to Figures 5, 6 , 8, 10 and 12 ). After this incubation, the alginate gel filter effect penetrates the targets 1 in the layer 2a until they are captured by the nanoparticles 2b. The latter have an average diameter of about 100 nm and can be functionalized or grafted with surface functions, for example of polystyrene sulfonate type.

Washing and de-gelling operations B of the alginate layer 2a were then carried out, which led to obtaining target-bound nanoparticles 2b in the alginate in solution, advantageously by means of a polycation-chelating agent which is, for example, for sodium polycations, ethylene diamine tetraacetic acid (EDTA) or sodium citrate.

Finally, a separation C has been carried out, advantageously by magnetization (the magnet used M is symbolized by a rectangle at the figure 1 ), nanoparticles 2b linked to the targets 1 which makes it possible to obtain, in view subsequent analyzes, such targets 1 as proteins adsorbed on the capture surface of nanoparticles 2b.

Polymeric composites 2 incorporating the nanoparticles 2b embedded in the porous polymeric layer 2a were prepared in the following manner. The nanoparticles 2b were first added and dispersed by stirring in an aqueous buffer composed of 154 mM NaCl and HEPES. Then a powdered alginate was added to the dispersion thus obtained in a mass fraction varying from 1% to 3%, and in particular equal to 1.5% for the two examples of the invention relating to figures 6 and 10 ), with rotary stirring and under ultrasound for at least 10 hours. In this way, the non-crosslinked composites 2 were obtained consisting of a polymerized alginate hydrogel 2a coating the nanoparticles 2b.

• dans l'exemple non conforme à l'invention de la figure 7, une armature 3 constituée d'un unique fil métallique de diamètre 140 µm (en acier inoxydable chirurgical AISI 316L) dans une aiguille 4 de diamètre extérieur égal à 0,8 mm,
• dans l'exemple selon l'invention de la figure 5, une armature 5 (dont seule la face radialement externe 5a est visible) formée d'une toile tissée ou tressée (en acier inoxydable chirurgical AISI 316L) globalement tubulaire présentant suivant un pas d'environ 50 µm des interstices 5b à travers lesquels va pénétrer le composite 2 pour revêtir en outre la face radialement interne de l'armature 5, dans une aiguille 6 de diamètre extérieur égal à 1,1 mm, et
• dans l'autre exemple selon l'invention des figures 8, 10, 12, une armature 5' microgrillagée (également en acier inoxydable chirurgical AISI 316L) dans une même aiguille 6, (les photographies des figures 10 et 12 permettent de mieux distinguer la structure micro-grillagée de cette armature 5', laquelle s'étend sur une surface 5'a et présente des ajours 5'b comme visible à la figure 8).

Then, independently of the non-gelled composites 2 thus prepared, the following are inserted:

• in the example not in accordance with the invention of the figure 7 an armature 3 consisting of a single wire 140 μm in diameter (surgical stainless steel AISI 316L) in a needle 4 with an external diameter equal to 0.8 mm,
• in the example according to the invention of the figure 5 , an armature 5 (of which only the radially outer face 5a is visible) formed of a generally tubular woven or braided fabric (in stainless steel AISI 316L) presenting, in a step of about 50 μm, interstices 5b through which will penetrate the composite 2 for further coating the radially inner face of the armature 5, in a needle 6 with an outside diameter equal to 1.1 mm, and
• in the other example according to the invention of Figures 8, 10 , 12 , a 5 'micro-grilled frame (also in surgical stainless steel AISI 316L) in the same needle 6, (the photographs of figures 10 and 12 make it possible to better distinguish the micro-grid structure of this frame 5 ', which extends over a surface 5'a and has openings 5'b as visible in FIG. figure 8 ).

In these three cases, the armatures 3, 5, 5 'are inscribed in a cylindrical volume of greater transverse dimension between 500 microns and 5 mm.

Each of these needles 4, 6 has been assembled in a syringe 7 (see FIG. figure 2 ) of 5 mL provided with a syringe driver device 8 and the syringe 7 to be gelled into the needle 4, 6 via this syringe 7, so that this composite 2 covers the corresponding reinforcement 3, 5, 5 '. After the needle 4, 6 filled with the composite 2, the syringe 7 was withdrawn and this needle 4, 6 was immersed in an aqueous gelling bath 9 based on polycations preferably calcium (16 mM NaCl, 20 mM CaCl ₂ ). As visible at figure 2 For example, the gelation of the composite 2 was carried out by dropwise addition with this gelling bath 9 contained in a bottle 10, and this was obtained after a relatively long time (preferably for at least three days) reticulated alginate gel beads 11 with a mean diameter of between 2 mm and 3 mm.

Composite "strands" 2 have been obtained covering the armature 3, 5, 5 'over part of the axial length thereof, this covering length being between 1 cm and 5 cm. The photographs of Figures 9 and 10 show these recoveries, it being specified that for a better visualization of the composite 2, these photographs were taken with the alginate layer 2a alone without the nanoparticles 2b which would have darkened the composite 2 by preventing to distinguish the armature 3, 5 '.

Thus, the armatures 5, 5 'having openings 5b, 5'b are partially embedded in the composite 2, the latter extending on either side of these armatures 5, 5' through these openings 5b, 5 ' b. The armature 5 delimits a tubular internal volume filled with composite 2, which extends beyond said internal volume through the openings 5b.

To carry out a sampling of targets 1 in the body fluid 12, the syringe 7 is assembled again on the needle 6 filled with composite 2 covering the armature 3, 5, 5 'in order to achieve the insertion of the needle 6 into the vein 12, as illustrated in FIG. figure 3 wherein the armature 5 and the composite 2 covering it have been shown schematically in the retracted position (ie below the open end of the needle 6). After the needle 6 has penetrated into the vein 12, the armature 5 covered with the composite 2 is pushed in translation along the needle 6, so that it opens out beyond the end of the latter in the position partially out of the figure 4 and that the nanoparticles 2b embedded in the composite 2 are in contact with the targets 1 contained in the vein 12. A liquid, preferably an injectable liquid, can be placed between the thrust means and the armature 5, 5 'so that to preserve the composite 2 during the push.

As illustrated in the photograph of the figure 6 it will be noted that the composite 2 (visible in gloss) covers the armatures 5, 5 'according to the invention, on the one hand, on their faces 5a, 5'a, but also on their faces situated on the other side of the armature. These frames 5, 5 'have a thickness of between 10 microns and 100 microns.

Comparative tests were carried out with the water washing of the composites 2 covering respectively the wire frame 3 of the Figures 7 and 9 , the frame 5 according to the Figures 5 and 6 and the armature 5 'according to the Figures 8 and 10 . For the washing of these three composites 2 was used a hand shower projecting deionized water ("DI" water) for the same duration of one minute.

The photographs of Figures 11 and 12 report the results obtained for these frames 3 and 5 '. As visible especially in the lower right portion of the figure 11 , the alginate gel has partially detached from the frame 3 not in accordance with the invention, while the figure 12 shows instead that the alginate gel of the composite 2 has withstood this wash and always covers the outer face of the armature 5 'to a uniform thickness over the entire axial length of the armature 5'. It is the same for the frame 5 of Figures 5 and 6 .

These comparative tests thus show that the risks of release of polymer fragments of the composite 2 into the body fluid are really minimized, thanks to the microporous frameworks 5, 5 'according to the invention.

It will be noted that the crosslinked composite 2, which covers the armature 5, 5 'according to the exemplary embodiments of the Figures 5, 6 , 8, 10 , 12 over part of its axial length, was obtained using the above-mentioned preferential mass fraction of 1.5% alginate in the aqueous dispersion. The viscosity of this crosslinked composite 2 consisting of the 1.5% alginate layer 2a in which the nanoparticles 2b are embedded was about 250 mPa.s (viscosity measured by a cone-plane rheometer), and its modulus of Young (calculated from compression tests carried out with a rheometer) was about 77 kPa, which contributes to the good adhesion of the composite 2 on the microstructured framework 5, 5 '.

Claims (15)

1. Sampling device (5 or 5', 2) adapted to be inserted into a hollow tubular endpiece (6) of the needle or catheter type, and to emerge from the endpiece with a view to contact, particularly in vivo, with a bodily fluid (12) containing biological targets (1) to be sampled, the device comprising:

   - a framework (5, 5') microstructured by openings (5b, 5'b), and

   - a biocompatible and porous crosslinked polymer layer (2a) which comprises capture supports (2b) adapted to capture the said targets and which is adapted to retain these supports from the fluid and to let through only fluid particles including these targets with a size of less than a cutoff size,
   the said polymer layer filling all or some of the said openings, so as to be retained by the said framework,
   characterized in that the said framework is substantially undeformable between positions in which it is inserted into the endpiece and in which it emerges from the latter.
2. Sampling device (5 or 5', 2) according to Claim 1, characterized in that the framework (5, 5') is circumscribed by at least one cylindrical surface having a largest transverse dimension of between 500 µm and 2 mm.
3. Sampling device (5 or 5', 2) according to Claim 1 or 2, characterized in that the framework (5, 5') has an external face (5a, 5'a) and delimits an internal volume, the said polymer layer (2a) extending through the said openings (5b, 5'b) from the said internal volume to the said external face and beyond the latter.
4. Sampling device (5 or 5', 2) according to one of Claims 1 to 3, characterized in that the said framework (5) has a single longitudinal symmetry axis which is intended to be parallel to that of the said endpiece (6), the said framework maintaining an overall tubular geometry in the said positions.
5. Sampling device (5 or 5', 2) according to one of the preceding claims, characterized in that the said crosslinked polymer layer (2a) has a viscosity, measured by a cone and plate rheometer, which is equal to or greater than 100 mPa.s and preferably lies between 150 mPa.s and 5800 mPa.s.
6. Sampling device (5 or 5', 2) according to one of the preceding claims, characterized in that the said framework (5, 5') is of the latticed, woven or plaited type, comprising a multitude of the said openings (5b, 5'b), separated in pairs by a pitch of between 30 µm and 60 µm,
7. Sampling device (5 or 5', 2) according to Claim 2 and one of Claims 3 to 6, characterized in that the said framework (5, 5') has a thickness of between 10 µm and 100 µm, the said at least one cylindrical surface having a substantially elliptical or circular cross section.
8. Sampling device (5 or 5', 2) according to Claim 3 and one of Claims 4 to 7, characterized in that the said external face (5a, 5'a) and/or an internal face of the framework (5, 5') furthermore has or have indentations and/or reliefs which are separate from the said openings (5b, 5'b) and which have dimensions of between 20 µm and 90 µm.
9. Sampling device (5 or 5', 2) according to Claim 3 and one of Claims 4 to 8, characterized in that the said polymer layer (2a) forms, with respect to the said external face (5a) of the framework (5, 5'), an external coating substantially coaxial with this framework and having a thickness of between 50 µm and 300 µm.
10. Sampling device (5 or 5', 2) according to one of the preceding claims, characterized in that the said crosslinked polymer layer (2a) has a Young's modulus, measured on the basis of compression tests carried out with a rheometer, of between 50 kPa and 270 kPa inclusive.
11. Sampling device (5 or 5', 2) according to one of the preceding claims, characterized in that the said framework (5, 5') is embedded in the said polymer layer (2a) over a part of its axial length lying between 1 mm and 5 cm, this polymer layer having a volume of between 1 ml and 10 ml.
12. Sampling device (5 or 5', 2) according to one of the preceding claims, characterized in that:

    - the said framework (5, 5') is made of metallic material, preferably stainless steel of surgical grade, silicon or a polymer material such as a silicone,

    - the said polymer layer (2a) is based on at least one biocompatible polymer with reversible gelling, selected from the group consisting of alginate gels, copolymers of alginate and poly-L-iysine, chitosan, agarose, cellulose, poly(trimethylammonium ethylacrylate methyl sulfate)-b-poly(acrylamide), poly(hydroxyethylmethacrylate (HEMA), poly(hydroxyethylmethacrylate-methyl methacrylate (HEMA-MMA) and other copolymers based on methacrylate, polyethylene glycols, copolymers of acrylonitrile and polyethylene glycol, polysaccharides and mixtures thereof, and in that

    - the framework is provided on its surface with functional groups creating chemical bonds between the framework and the polymer layer, preferably carboxylic acid or amine groups in the case in which the framework is metallic for bonding with hydroxyl groups of this layer.
13. Sampling system (6, 7, 5 or 5', 2) comprising:

    - a hollow tubular endpiece (6) of the needle or catheter type, which has an internal diameter of between 500 µm and 2 mm,

    - a sampling device (5 or 5', 2) inserted into the endpiece and capable of emerging by sliding from an end of this endpiece with a view to contact, particularly in vivo, with a bodily fluid (12) containing biological targets (1) to be sampled, and

    - a thrust member (7) capable of making the said sampling device slide reversibly out of the said endpiece,
    characterized in that this device is as defined in one of the preceding claims, this device optionally being provided with a means for connection to the said end of the endpiece, and preferably in that the said thrust member (7) is of the syringe type and comprises:

    - a pump body, in which the endpiece (6) is mounted, and

    - a rod which can be inserted into the said endpiece in order to make the said sampling device (5 or 5', 2) slide therein.
14. Method for manufacturing a sampling device (5 or 5', 2) according to one of Claims 1 to 12, characterized in that it comprises the following steps:

    a) preparation of an uncrosslinked polymer composite (2) incorporating the said capture supports (2b) and the said uncrosslinked polymer layer (2a) covering them,

    b) insertion of the said framework (5, 5'), without this composite, into a tubular mold, optionally with connection of the framework to a sampling end of the endpiece,

    c) assembly of the endpiece containing this framework in a sampling member (7) of the syringe type,

    d) take-up of the uncrosslinked composite prepared in a) by this sampling member, in order to inject this composite inside the endpiece in contact with the framework, then

    e) crosslinking in a gelling bath of the endpiece which is filled with the uncrosslinked composite injected in d) and which has previously been extracted from this sampling member, in order to obtain the said crosslinked polymer layer fixed to the framework.
15. Manufacturing method according to Claim 15, characterized in that step a) comprises:

    a1) dispersion in an aqueous buffer solution of the said capture supports (2b) comprising magnetic or non-magnetic functionalized nanoparticles, then

    a2) addition under agitation to the dispersion obtained in a1) of at least one biocompatible polymer with reversible gelling, in order to obtain the said uncrosslinked composite (2) in which these nanoparticles are embedded.

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